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Bioseparations

2D gel
© PIXANIM@INRAE
PIXANIM offers 1D/2D electrophoresis and liquid chromatography for the separation and purification of proteins.

Protein extraction

Qualitative and quantitative proteomics requires the use of quality biological samples. The extraction of proteins from organs, tissues, isolated cells or physiological fluids is carried out using appropriate buffers according to the nature of the proteins to be studied (cytosolic, membrane, nuclear proteins, etc.). Extraction buffers at a given pH are composed of mixtures of reducing agents, detergents or even organic solvents. They are generally supplemented with protease inhibitors. Experimental protocols must avoid contamination by nucleic acids, lipids and salts. In general, the protocols must be adapted for each biological model.

2D/1D electrophoresis

2D electrophoresis enables the proteins in a complex mixture to be separated in two stages according to two distinct properties of these molecules: their charge and their relative molecular mass. The first dimension consists of depositing the protein mixture on a polyacrylamide gel containing a stable pH gradient (carrier ampholytes or immobilised gradient). Isoelectrofocusing (IEF) consists of subjecting the proteins to an electic field.
The proteins, amphiphilic molecules (which carry positive and negative charges located on the side chains of the amino acids) are then separated according to their isoelectric point (pI). They migrate until their electric charge is zero. Different types of immobilised pH gradients allow proteins to be separated in the 1st dimension in the extreme acidic or basic pH ranges, or over the whole pH range 3-12. Narrow, overlapping pH gradients break down the pH range 4-7 into a single pH unit (4-5, 5-6, etc.). The number of proteins detected is then doubled. The narrow gradients between pH 7 and 10 remain difficult to use.
Before the 2nd dimension, the IEF gels are re-balanced in order to impregnate the gel with SDS (sodium dodecyl sulphate), a strong acid denaturing detergent, which will give the proteins a negative charge. The SDS interacts with the proteins and gives each one the same charge/mass ratio. Once the IEF gel is saturated with SDS, it is deposited on top of an acrylamide gel so that the molecules are separated according to their apparent mass (kDa). The protein stains are then revealed by different colourings.
Hydrophobic proteins such as membrane proteins are difficult to solubilise. Similarly, very basic proteins, such as histones and ribosomal proteins, are hardly visible on a 2D gel after isoelectrofocusing.
Some classes of proteins encounter some analytical problems with 2D electrophoresis. In particular, two critical steps have been identified: the first dimension of separation (IEF) and transfer to the second dimension. In order to avoid losses of material at these stages, an alternative consists in simply separating the protein mixture of interest by mono-dimensional electrophoresis on acrylamide gel, SDS-PAGE. In the case of complex protein mixtures, a coloured gel strip may contain many proteins. The identification of the proteins present in a band then requires nanoLC-MS/MS (cf bottom-up proteomics).

Liquid chromatography

Chromatography is an analytical technique allowing the separation of the constituents of a mixture, based on the distribution of these constituents between a mobile phase (liquid or gas) and a stationary phase (solid or liquid) with a large contact surface. Liquid-solid chromatography is well suited to the separation of amphiphilic ionised macromolecules such as proteins.